The work in this proposal is focused on a major cancer-preventive signal transduction pathway that triggers transcriptional induction of enzymes that protect cells from reactive chemical species, including carcinogens and oxidative stress. This pathway is activated by both naturally occuring chemopreventive agents found in a wide variety of fruits and vegetables and by synthetic molecules. We believe that a fundamental understanding of this signal transduction pathway will facilitate the identification of foods, dietary supplements and drugs that will significantly decrease the risk of cancer in humans. Furthermore, as oxidative stress is a driving force of many pathophysiological conditions, including neurodegeneration, cardiovascular disease and skeletal muscle atrophy,the proposed research will have a broad impact on human health. The critical target of this pathway, the transcription factor Nrf2, is normally repressed by the BTB-Kelch protein, Keapl. Chemopreventive agents enable Nrf2 to escape Keapl-mediated repression andactivate transcription of its target genes that eliminate reactive species and restore cellular redox homeostasis. Our preliminary data suggest the hypothesis that Keapl functions as a substrate adaptor protein for a Cul3- dependent E3 ubiquitin ligase complex. This hypothesis represents a new paradigm for understanding how Keapl is able to repress Nrf2-dependent transcription and provides a productive framework for defining how chemopreventive agents enable Nrf2 to escape Keapl-mediated repression. This hypothesis also provides novel insight into the biological functions of all BTB-Kelch proteins. We propose to examine this hypothesis further by characterizing disease-associated mutations within GAN1 and ENC1 that are responsible for giant axonal neuropathy and contribute to brain cancers, respectively. The proposed experiments will (1) define how Nrf2 is targeted for ubiquitin-dependent degradation by a Keapl :Cul3:Rbx1 complex, (2) define how Nrf2 escapes Keapl-mediated repression, (3) use Keapl as a model system to define how disease-associated mutations perturb the substrate adaptor function of BTB- Kelch proteins, and (4) define the structural basis for substrate recognition by Keapl.